ENVIRONMENTAL SCIENCE AND ENGINEERING PROGRAM
Transcript of ENVIRONMENTAL SCIENCE AND ENGINEERING PROGRAM
ENVIRONMENTAL SCIENCE AND ENGINEERING PROGRAM
Environmental Analytical Chemistry
I. Principles of Mass Spectrometry
Environmental Chemical Processes Laboratory
Euripides G. Stephanou
HistoryModern mass spectrometer: • J. J. Thomson (1906 Nobel Prize in Physics)
Higher accuracy mass spectrometers: • F. W. Aston (1920 Nobel Prize in Chemistry), A. J. Dempster
Advances in vacuum technology and electronics:• A. Neir
Time-of-flight analyzers:• Wiley and McLaren 1955
Quadrupole analyzer:• W. Paul (1989 Nobel Prize in Physics)Electrospray ionization • J. Fenn (2001 Nobel Prize in Chemistry), M. Dole
Matrix assisted laser desorption/ionization (MALDI)• Tanaka (2001 Nobel Prize in Chemistry)
Mass spectrometry can be defined as an instrumental approach that allows for the mass measurement of molecules in very low quantities
(as low as 100 * 10-18 moles). The five basic parts of any mass spectrometer are:
5) An ion detector.
1) A vacuum system. 2) A sample introduction device.
3) An ionization system (A⇒A+). 4) A mass analyzer (m/z).
Theory and Practice of Mass Spectrometry
1. Vacuum System Mass spectrometry requires a low pressure to operate.
• Low vacuum measurement• Operates on principle of the Wheatstone bridge (Rg is a thermistor)
• It uses the thermal conductivity of gases to measure pressure.
• The system is pumped down: there are less molecules and therefore less collisions. Fewer collisions mean that less heat is removed from the wire and so it heats up. As it heats up its electrical resistance increases. A simple circuit utilising the wire detects the change in resistance and once calibrated can directly correlate the relationship between pressure and resistance.
Pirani or Convectron™ Gauge
Ion Gauge
• High vacuum measurement• Outgasing used to dispel contaminants• Ions are formed at the filament and attracted toward collector• Ions striking grid generate current
Gauge Pressure Range
Typical Use
Manometer 760 - 1 torr systems near atmospheric pressure
Thermocouple gauge
1 - 10-3 torr monitoring mechanical pumps
Ionization gauge 10-3 - 10-9 torr high-vacuum systems
Common pressure gauges
Common vacuum pumps Pump Lowest Attainable
Pressure Typical Use
Mechanical pump 10-3 - 10-4 torr roughing or backing pump
Diffusion pump 10-6 torr vacuum linesTurbomolecular pump
10-9 torr high-vacuum systems
Sample Introduction Device
The sample inlet is the interface between the sample and the mass spectrometer
To introduce pure compounds: samples are placed on a probe which is then inserted
through a vacuum lock into the ionization source
Gas chromatography (GC)
Capillaries are used to interface the ionization source with other separation techniques:
Liquid chromatography (LC)
Mol
ecul
ar W
eigh
t
Non-Polar Polar
EI /
CI
Electrospray
APCI
100,000
1000
Which Ionisation Mode ?
Ionization Techniques
Electron Ionization – Electron Impact (EI)
Electrons are produced by thermionic emission from a tungsten or rhenium filament (filament current ca. 1*10-4Amps).
e- energy: 70 eV
+
Mechanisms of ion formation in EI sources
AB + e-* -----> A+ + B- + e-
AB + e-* -----> A+ + B° + 2e-
AB + e-* -----> [AB+°*] + 2e-
and [AB+°*] -----> AB+°
AB + e-* -----> [AB2+*]" + 3e-
and [AB2+*]" -----> A+ + B+
AH + e-* -----> AH* + e- and
AH* + AH -----> [AH+H]+ + A-
High abundance of ions
Low abundance:Molecular ion
Formation
Very lowabundance but
possible
'Self chemical ionization'
Chemical ionization (CI)A reagent gas is ionized at a pressure of 0.3-1 torr to produce a high yield of reagent ions which may be positively or negatively charged and react with the molecules to form ions which constitute the CI spectrum of the compound.
I) Acid-base reaction type:
M (molecule of sample) + XH+ (reagent ions) --> MH+ (“molecular ions”)+ X (reagent
gas)
II) Complex formation reaction type:
M (molecule of sample) + XH+ (reagent ions) --> MXH+ (“molecular ions”)
III) Charge transfer reaction type (redox):
M (molecule of sample) + G+• (reagent ions) --> M+• (“molecular ions”)+ G (reagent
gas)
The reagent gases in positive CIThe most utilized reagent gases are methane (CH4), ammonia (NH3), isobutane (i-C4H10) and noble gases for the charge transfer reactions.
CH4 (EI ionization) --> CH4+ , CH3+ , CH2+ , CH+ , C +
CH4+ + CH4+• --> CH5+ (47%) + CH3
CH3+ + CH4 --> C2H5+ (41%) + H2
CH4 + C2H3+ --> C3H5+ (6%) + H2
0
5
10
15
20
25
30
35
40
45
50
17 29 41
mass/charge
Ion
Abun
danc
e (%
)
CH5+ + M --> MH+ + CH4↑ ( [m/z]+1)
C2H5+ + M --> MH+ + C2H4↑ ( [m/z]+1)
C2H5+ + M --> [M C2H5]+ ( [m/z]+29)
C3H5+ + M --> [M C3H5]+ ( [m/z]+41)
"Molecular ions" in Methane CI
MW+1
MW+29
MW+41
0
5
10
15
20
25
m/z
rela
tive
abun
danc
e (%
)Methane chemical ionization
M2-M1=28 amuM3-M1=40 amuM3-M2=12 amu
⇓M1-1=MW
NH3 (EI ionization) --> NH3+•
NH3 + NH3+• --> NH4+ + NH2•
NH3 + NH4+ --> (NH3)2H+
The reagent ions react with the molecules like Brønsted or like Lewis acids:
NH4+ + M --> MH+ + NH3↑ ( [m/z]+1)
NH4+ + M --> [M NH4]+ ( [m/z]+18)
Ammonia chemical ionization
Mass Spectrometric Study in Chemical Ionisation
EI
NH3 isoboutane
Methane
Nopinone (Ret. Time 4.05) MW=138
M+•
[M+H]+
[M+H]+
[M+H]+
[M+C2H5]+
[M+C3H5]+
[M+NH4]+
Electrospray, Microspray, Nanospray Ionisation
Atmospheric PressureChemical Ionisation
Atmospheric Pressure (API) Ion Generation
TSQ Quantum Discovery API Probes
Electrospray APCI
Adjustable Angle ES Source Fixed orthogonal APCI source
Adjustable Angle Electrospray Geometry Optimal LC flow capability (50 µl/min – 800 µl/min)
Accepts fused silica or metal needle
Max voltage 8 kV, 100 µA
Electrospray Probe
Electrospray Ionization (ESI)
The solvent evaporates away, shrinking the droplet size and increasing the charge concentration at the droplet's surface.
The charges are statistically distributed: formation of multiply charged ions.
Large charged droplets are produced by forcing of the analyte solution through a needle, at the end of which is applied a potential (e.g. 4,000 V)The emerging solution is dispersed into a very fine spray of charged droplets of the same polarity.
Multiply Charged Ions- Mass Assignments Single Charge - apparent mass = (M+H)/1 Double Charge - apparent mass = (M+2H)/2 Triple Charge - apparent mass = (M+3H)/3 n charges - apparent mass = (M+nH)/n
100
Substance P
670 671 672 673 674 675 676 677 678 679 680 Da/e 0
%
674.6
675.1
675.6
676.1 676.5
The isotopes of doubly charged ions are separated by 0.5 amu The isotopes of triply charged ions are separated by 0.33 amu
Charge State is ?
APCI Probe: Atmospheric Pressure Chemical Ionization The liquid effluent is introduced directly into the APCI source APCI source contains a heated vaporizer: rapid vaporizationIonization occurs through a corona discharge: reagent ions from the solvent vapor Vaporized molecules carried through ion-molecule reaction at atmospheric pressure.
Heated nebuliserSample moleculesSolvent molecules
Liquid+
+
+ +
+
Corona pin
Aerosolforms
Sample molecules solvated
Solventionised
Sampleionised
Charge transfer
+
N2
N2 +
Mechanism of ion generation in APCI
Protonation (MH+) occurs in the positive mode, and e- transfer or H+ loss ( [M-H]-) in the negative mode.
Chemical ionization of sample molecules is very efficient at atmospheric pressure (high collision frequency)
Primary ion formation:
Secondary ion formation:
Positive analyte ion formation:
Negative analyte ion formation:
H2O + e- H2O+• + 2e-
H2O+• + H2O H3O+ + • OH
H3O+ + Analyte [Analyte+H]+ + H2O
• OH + Analyte [Analyte-H]- + H2O+.
APCI Reaction Mechanism
ESI:Ions are predominantly pre-formed in solutionTechnique works well with Polar analytes
Good for Thermally labile analytesGood for Large Molecules (Proteins/Peptides)
APCI:Ions are formed by gas phase chemistryTechnique works well with Non-Polar analytes Good for Volatile / Thermally Stable analytesGood for Small Molecules (Steroids)
Chemistry Considerations
Ionization Analytes Introduction
Max mass
Capability
EIRelatively small, volatile
GC or probe
1,000 Daltons
Provides structure info
CIRelatively small, volatile
GC or probe
1,000 Daltons
Molecular ion [M+H]+
ESIPeptides, Proteins, Nonvolatile
LC or syringe
200,000 Daltons
Ions often multiply charged
1) Double Focusing (Sector) Analysis
scanned fixed
Increase in resolution allows for better peak distinction within a spectrum
Solely magnetic sector Magnetic+electric sectors
3) Quadrupole Mass Analyzer Electric fields are used to separate ions according to their m/z values
-(U+Vcos(ωt)) +(U+Vcos(ωt))
m/z = ƒ (U,V,ω)
Choice of U, V and ω (1-2 MHz): only one m/z will oscillate stably through the quadrupole mass analyser to the detector
Full Scan: As RF and DC voltages are ramped upward (i.e.the mass analyzer is scanned), ions of successively higher mass-to-charge ratios and having stable trajectories are allowed to pass through the analyzer. If one MS scan between m/z 0 and m/z 500 is completed in one second, then each m/z will be allowed to pass for only 1/500 s.
Full Scan v’s SIM
SIM: If the RF and DC voltages are held constant, ions of a single m/z ratio and having a stable trajectories are transmitted. Data is collected on the ion for a much longer time resulting in improved signal to noise, better peak definition and lower RSD’s. SIM can give 15 to 25 times improvement in sensitivity compared to full scan.
+
+
+ +
5) Ion Trap Analysis
• Voltages are applied to the 3 electrodes to trap and eject ions according to m/z. • The ring electrode RF potential, is producing a 3D quadrupolar potential field within the trapping cavity. • Ions are trapped in a stable oscillating trajectory. • The trajectory depends on the trapping potential and the m/z. • Alteration of potentials: Instabilities in trajectory: Ions are ejected in increasing m/z and focused by the exit lens and detected by the ion detector system.
2) Time-of-Flight (TOF)
E = extraction field=Vs/lsti = time-of-flight of ionls = length of the sourceld = length of the field-free drift regione = electronic charge
Source extractionpotential
Quadrupoles Ion traps
and TOF’s
Constant peak widths
Variable Resolution
Resolution FWHM: Full Width Height Maximum
0.7 FWHM
m/z 300/ 0.7 = R 428m/z 500/ 0.7 = R 714
m/z 1000/ 0.7 = R 1428
0.7 u
“Unit mass Resolution” v’s 0.1 FWHM
m/z 300/ 0.1 = R 3000m/z 500/ 0.1 = R 5000
m/z 1000/ 0.1 = R 10,000
0.1 u
0.1 FWHM
6) Tandem Mass Spectrometry
Characteristic Precursor ions
MS spectrum
MS –MS spectrum Product ions
• Isolation of the precursor ion • Fragmentation of the precursor ion • Detection of product ions
MSn
Fragmentation: induction of ion/molecule collisions by collision-induced dissociation (CID).
Q1
Q3
Q2
Detection System
ES Source
TSQ Quantum Components
Ion Optics
Analyser
Analyser
Collision Cell
Electron Multiplier
Conversion Dynode
Ion Trap “MS-MS”
Fragmentation: induction of ion/molecule collisions by collision-induced dissociation (CID) within the ion trap.
Analyzer System capabilities
Quadrupole Unit mass resolution, fast scan, low cost
Magnetic and/or Electrostatic High resolution, exact mass
Time-of-Flight (TOF)
Theoretically, no limitation for m/z maximum, high throughput
Overview of the most used mass analyzers
5. Ion Detection
Faraday cup
Electron multiplier
Photomultiplier conversion dynode
Applications of Mass Spectrometry
• Atomic Masses• Geochronology and Geochemistry
• Accelerator MS and dating materials• Organic Chemistry
• Combinatorial Chemistry • Biochemistry
• Peptide and DNA sequencing• Small biomolecule characterization
• Viruses• Forensics
• Space probes• Small Mass Spectrometers
Element Isotope Relative Abundance Isotope Relative
Abundance Isotope Relative Abundance
Carbon 12C 100 13C 1.11 Hydrogen 1H 100 2H .016 Nitrogen 14N 100 15N .38 Oxygen 16O 100 17O .04 18O .20Sulfur 32S 100 33S .78 34S 4.40Chlorine 35Cl 100 37Cl 32.5Bromine 79Br 100 81Br 98.0
Common isotopes of the most important elements
Methyl Bromide: An example of how isotopes can aid in peak identification.
The ratio of peaks containing 79Br and its isotope 81Br (100/98) confirms the presence of bromine in the compound
Common Mass Spectrum Fragments
Stages of a mass spectrum interpretation-1:
• 1. Look for the molecular ion peak:
• This peak (if it appears) will be the highest mass peak in the spectrum, except for isotope peaks.
• Nominal MW will be an even number for compounds
containing only C, H, O, S, Si. • Nominal MW will be an odd number if the compound
also contains an odd number of N (1,3,...).
Stages of a mass spectrum interpretation-2:
• 2. Calculate the molecular formula: The isotope peaks can be very useful, and are best
explained with an example. • 12C has an isotope of 13C. Their abundances are
12C=100%, 13C=1.1%. This means that for every 100 12C atoms there are 1.1 13C atoms.
• Example: If a compound contains 6 carbons, then each
atom has a 1.1% abundance of 13C.
If the molecular ion peak is 100%, then the isotope peak (1 mass unit higher) would be 6x1.1%=6.6%.
Stages of a mass spectrum interpretation-3:
• If the molecular ion peak is not 100% then calculate the relative abundance of the isotope peak to the ion peak.
• Example: if the molecular ion peak were 34% and the isotope peak 2.3%:
(2.3/34)x100 = 6.8%. 6.8% is the relative abundance of the isotope peak to the ion peak.
Next, divide the relative abundance by the isotope abundance: 6.8/1.1=6 carbons.
• Look for A+2 elements: O, Si, S, Cl, Br• Look for A+1 elements: C, N• "A" elements: H, F, P, I
Stages of a mass spectrum interpretation-4:
• 3. Calculate the total number of rings plus double bonds:
• For the molecular formula: CxHyNzOn • Rings + Double Bonds = x - (1/2)y + (1/2)z + 1
• 4. Postulate the molecular structure consistent with abundance and m/z of fragments.
More information on specific fragmentation can be found for each functional group.
Example #1 Analysis: C5H12O MW = 88.15
[M]+•
[M-H]+ [M-CH3]+
[M-H2O]+
[M-C3H7]+
[M]+• [M-H]+
[M-CH3]+ [M-H2O]+
[M-C3H7]+
Example #2 Analysis: C7H12Br MW = 171.04
[M]+• [M+2]+•
Tropylium ion
[M-Br]+
[Tropylium –C2H2]+
Example #3Analysis: C9H10O MW = 134.18
[M]+• [M-15]+
Tropylium ion
[Tropylium –C2H2]+
[M-91]+
Example #4Analysis: C11H12O 3 MW = 192.21
[M]+• [M-45]+
[M-87]+
[C6H5]+
or [C6H5-CO]+
Example #5Analysis: C5H8O2 MW = 100.12
[M]+•
[M-43]+
[M-57]+
Interpreting Electrospray Mass Spectra
The Y axis is labeled relative intensity.
The X axis is mass divided by charge, m/z
Is the "base peak"
The spectrum has a certain number of counts associated with the tallest peak in the spectrum.
All of the peaks in a spectrum should not be referred to as ions.
If the mass of a moleculeis 2000 and during ESI is charged with 2 H+ ⇒ (2000+2)/2=1001
If the mass is 2000 and is charged with 1 H+ ⇒ (2000+1)/1=2001
Calculating Mass Determining the charge state of the peaks:
Two adjacent peaks are from the same compound and differ by only one charge
190.1 = ma/z a 379.2 = mb/zb
m a= 190.1 (za) m b= 379.2 (zb)
If the 2 peaks differ by one charge:ma=m+1=190.1(z+1) mb=m = 379.2(z)
m assumed the same for both peaks m = [190.1(z + 1)] - 1
190.1 z + 189.1 = 379.2 zz = 1
Two peaks in the spectrum are mathematically related : • the charge state of the 379.2 peak is +1 • the charge state for the 190.1 peak is +2
Charge Calculation Unprotonated Mass+1 (379.2 - 1)*1 = 378.2+2 (190.1 - 1) *2 = 378.2
average 378.2